In tumors, loss of function of either p53 itself (Hollstein et al. Nucleic Acids Res. 1996 24:141-146; Nigro et al. Nature 1989 342:705-708), or of the p53 dependent pathway that activates G1 arrest, is one of the major and most frequent molecular events (reviewed in Sherr, C. J. Genes Dev. 1998 12:2984-2991). p53 function may be compromised directly, via genetic mutation and/or deletion of the p53 gene (Baker et al. Science 1989 244:217-221) and indirectly by changes in the regulation or level of the MDM2 protein (Oliner et al. Nature 1992 358:80-83). The MDM2 gene, itself a transcriptional target of p53 (Barak et al. EMBO J 1993 12:461-468; Juven et al. Oncogene 1993 8:3411-3416; and Wu et al. Genes Dev. 1993 7:1126-1132), encodes a protein, MDM2, that is a critical negative regulator of p53 function (Finlay, C. A. Mol. Cell. Biol. 1993 13:301-306; Momand et al. Cell 1992 69:1237-1245). MDM2 was originally discovered as an oncogene that was amplified on mouse double minute chromosomes (Cahilly-Snyder et al. Cell Mol. Genet. 1987 13:235-244). MDM2 was later found to be amplified and overexpressed in a variety of human cancers (Ladanyi et al. Cancer Res. 1993 1:16-18; Reifenberger et al. Cancer Res. 1993 53:2736-2739). MDM2 binds to the transcriptional activation domain of p53 and thus inhibits this function of p53 (Chen et al. Mol. Cell Biol. 1993 13:4107-4114; Oliner et al. Nature 1993 362:857-860). Moreover, MDM2 binding to p53 regulates the stability of the p53 protein such that p53 is ubiquitinated and is then degraded by the proteasome (Haupt et al. EMBO J. 1996 15:1596-1606; Kubbutat et al. Nature 1997 387:299-303). This, together with the observed effect upon p53 function, has led to a model in which an autoregulatory loop connects MDM2 and p53 (Barak et al. EMBO J 1993 12:461-468; Wu et al. Genes Dev. 1993 7:1126-1132).
MDM2 inhibits both p53 mediated G1 arrest and apoptosis (Chen et al. Mol. Cell. Biol. 1996 16:2445-2452; Haupt et al. EMBO J 1996 15:1596-1606). p53 induces G1 arrest by promoting transcriptional upregulation of the CDK inhibitor p21waf1/cip1 (Waldmann et al. Cancer Res. 1995 55:5187-5190). Therefore, it is likely that MDM2 prevents p53 from inducing G1 arrest by inhibiting p53 dependent transcriptional activation. MDM2 can prevent p53-mediated apoptosis, and this has been shown to be dependent upon the ability of MDM2 to inhibit transcriptional repression by p53 (Hsieh et al. Mol. Cell 1999 3:81-93). Moreover, a previously identified interaction with RB (Xiao et al. Nature 1995 375:694-698) was shown to be able to regulate this effect. By binding to MDM2, RB forms a stable ternary complex with p53 and this prevents the MDM2 promoted degradation of p53. The ternary complex can promote p53 dependent apoptosis but not p53 mediated transactivation.
The autoregulatory relationship between p53 and MDM2 suggests that MDM2 overexpression may be oncogenic because of the resulting inactivation of p53 (Wu et al. Genes Dev. 1993 7:1126-1132). This conclusion is supported by studies of human tumors which show that in the majority of cases either p53 is mutated/deleted or MDM2 is overexpressed (Leach et al. Cancer Res. 1993 53:2231-2234). Studies of allelic knockouts of these genes in mice further support the ability to negatively regulate p53 being a primary function of MDM2. Mice that possess a homozygous deletion of MDM2 die at around day 5 of embryogenesis whereas, mice that possess homozygous deletion of both MDM2 and p53 are viable and develop normally (Jones et al. Nature 1995 378:206-208; Montes de Oca Luna et al. Nature 1995 378:203-206). No differences have been detected between these p53 −/− and p53−/−, MDM2−/− mice in terms of the rate or spectrum of tumors developed (Jones et al. Proc. Natl Acad. Sci. USA 1996 93:14106-14111). Also, no differences could be detected between the embryonic fibroblasts derived from these animals in terms of their growth or cell cycle characteristics.
Collectively, these observations suggest that the primary function of MDM2 may be to regulate p53 activity and perhaps, during normal development, this is indeed the case. However, the situation appears to be different when MDM2 is expressed at abnormally high levels. Experiments in which MDM2 was overexpressed in NIH3T3 cells have shown that naturally occurring splice variants of MDM2 that lack the ability to bind to p53 are still able to transform these cells (Sigala et al. Nat. Med. 1996 2:912-917). Further support for the suggestion that MDM2 has p53 independent effects is derived from studies of transgenic mice. Mice transgenic for an MDM2 gene expressed from a β-lactoglobulin promoter, exhibited abnormal mammary development, with cells becoming polyploid together with a multinucleate morphology, suggestive of DNA synthesis in the absence of mitosis (Lundgren et al. Genes Dev. 1997 11:714-725). The same results were obtained in both p53 wild type animals and in animals with homozygous deletion of p53. In addition, recent studies using a different transgenic system with multiple copies of the whole MDM2 gene being used to generate mice that overexpress MDM2 from the MDM2 promoter, have shown that these animals develop a different spectrum of tumors c.f. p53 null mice (Jones et al. Proc. Natl Acad. Sci. USA 1998 95:15608-15612). The same effect of MDM2 overexpression was observed regardless of the p53 status of these animals. Finally, in support of the existence of p53 independent effects of MDM2 upon overexpression, it has recently been shown that MDM2 has the ability to abrogate the growth inhibitory activities of Transforming Growth Factor-Betal (TGFβ1). This effect was p53 independent in cells in culture (Sun et al. Science 1998 282:2270-2272). Taken together, these results all suggest that overexpression of MDM2 acts not only upon p53 but also on additional pathways.
Using a yeast two hybrid screen a novel gene encoding a protein referred to herein as MTBP for MDM2 (Two) Binding Protein has now been identified. MTBP is demonstrated herein to be capable of negatively regulating growth by inducing G1 arrest in a p53 independent manner. Further, this negative regulation of growth can be suppressed by MDM2.